Retroviral infections often spread by cell-to-cell transmission, which involves transfer of viruses from infected cells to neighboring uninfected cells via zones of cell-cell contact designated virological synapses. Our laboratory has contributed to the understanding of the biogenesis of virological synapses using a model retrovirus, the Friend murine leukemia virus (F-MLV). Interactions between the viral envelope glycoprotein (Env) in the infected cell and its viral receptor in the target cell drive the establishment of cell-cell contact. The viral Env accumulates in the cell-cell interface and redirects virus assembly to the site of cell-cell contact. Our work has, for the first time, documented the transmission of viruses from one cell to another using time-lapse video microscopy and shown that polarized assembly directly at the cell-cell interface can contribute to the efficient spreading of retroviral infections. While we have made progress in understanding the biogenesis of virological synapses using tissue culture systems, little is known about the mechanism of virus spreading in vivo. To bridge this gap in knowledge, we have established intravital microscopy to visualize the behavior of infected lymphocytes directly in vivo within the lymph node of living mice. We identify a subpopulation of F- MLV-infected B cells that form virological synapses with uninfected lymphocytes. In vivo virological synapses are dependent on the viral envelope glycoprotein and are characterized by a prolonged polarization of viral capsid to the cell-cell interface. Our results suggest that Env-induced virological synapses exist in vivo and can contribute to the spreading of retroviral infections. In this competitive renewal application we will study MLV cell-to-cell transmission in relevant primary lymphocytes. We will explore the role of infected B and T cells in the in vivo spread of MLV. We will elucidate the mechanism of polarized virus assembly in in vivo virological synapses and will identify viral and cellular factors that synergize with the Env-receptor interactions to promote viral spreading to CD4+ T cells. We will also test if migratory T cells contribute to viral spreading by a mechanism that involves more transient cell-cell interactions. Our work will allow critical insights into pathogenesis and the spreading of retroviral infections.